Comparative Mapping in thebeige–satinRegion of Mouse Chromosome 13

GENOMICS

39, 136–146 (1997) GE964478

ARTICLE NO.

Comparative Mapping in the beige–satin Region of Mouse Chromosome 13 C. M. PEROU,* A. PERCHELLET,† T. JAGO,† R. PRYOR,* J. KAPLAN,*

AND

M. J. JUSTICE†,1

*Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132; and †Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received August 6, 1996; accepted October 30, 1996

The proximal end of mouse chromosome (Chr) 13 contains regions conserved on human chromosomes 1q42–q44, 6p23–p21, and 7p22–p13. This region also contains mutations that may be models for human disease, including beige (human Chediak–Higashi syndrome). An interspecific backcross of SB/Le and Mus spretus mice was used to generate a molecular genetic linkage map of mouse chromosome 13 with an emphasis on the proximal region including beige (bg) and satin (sa). This map provides the gene order of the two phenotypic markers bg and sa relative to restriction fragment length polymorphisms and simple sequence length polymorphisms in 131 backcross animals. In parallel, we have created a physical map of the region using Nidogen (Nid) as a molecular starting point for cloning a YAC contig that was used to identify the beige gene. The physical map provides the fine-structure order of genes and anonymous DNA fragments that was not resolved by the genetic linkage mapping. The results show that the bg region of mouse Chr 13 is highly conserved on human Chr 1q42–q44 and provide a starting point for a complete functional analysis of the entire bg–sa interval. q 1997 Academic Press

INTRODUCTION

Several regions of the mouse genome are characterized by extensive linkage homology to a single human chromosome. The best example is the extensive conservation of human chromosome (Chr) 17 on mouse Chr 11 (Lossie et al., 1994). However, other regions of the mouse genome are more fragmented with respect to human homologies and contain regions conserved on multiple human chromosomes. In these regions, it is important to clarify the extent of linkage homology to extend and confirm syntenic groups of genes. The proximal end of mouse Chr 13 is such a region, with two linkage groups conserved on human Chr’s 7p and 6q, and two loci mapping to human Chr 1q42–q44 (Justice and Stephenson, 1996). In the process of identifying 1

To whom correspondence should be addressed.

0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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the murine beige gene (Perou et al., 1996b), we also further defined the mouse and human linkage relationships on the distal end of human Chr 1q. The inbred strain of mice SB/Le is homozygous for the mutations beige (bg) and satin (sa). Homozygous bg/bg mice exhibit partial albinism (Russell 1957), increased bleeding times (Swank et al., 1984), and an increased susceptibility to infections (Lane and Murphy, 1972). The beige mouse is an animal model for the autosomal recessive disorder in humans called Chediak–Higashi syndrome (CHS) (Holcombe et al., 1987; Perou and Kaplan, 1993). The similar clinical symptoms observed in CHS patients and bg/bg mice are due to a malregulation of vesicle fusion or fission that results in the formation of ‘‘giant’’ vesicles, particularly lysosomes. It is thought that the formation of these giant vesicles causes all of the phenotypes seen in these disorders. These giant vesicles are clustered near the nucleus of cells and cannot move throughout the cytoplasm, causing impaired degranulation (Perou and Kaplan, 1993). Because of these above-mentioned defects, basic cellular function, as well as immune cell function, is greatly impaired, causing afflicted CHS individuals or homozygous beige mice to be susceptible to infections. The sa locus is identified by a single allele that causes a shiny coat in mice (Russell, 1955) and that has no known human homologue. The defect in immune cell function observed in homozygous bg/bg mice is worse in double homozygotes with sa/sa (McGarry et al., 1984). Although bg and sa are interesting in their own right, other loci that represent human disorders also map to the bg–sa interval, including the murine homologue of human stiff man syndrome (amphiphysin) (Jenkins et al., 1995), Greig cephalopolysyndactly syndrome (mouse Extra-toes/Gli3) (Brueton et al., 1988; Winter, 1988), the T-cell receptor gamma complex (Murre et al., 1985), and genes predisposing individuals to cancer (Sola et al., 1988). An interspecific backcross between SB/Le and Mus spretus mice was used to order the bg and sa mutations relative to gene markers and anonymous DNA markers as a first step in genetically defining this interval (Jen-

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kins et al., 1991; Justice et al., 1990). In parallel to the genetic mapping, we have derived a 2.5-megabase (Mb) yeast artificial chromosome (YAC) contig in the bg region using Nidogen (Nid) as the molecular entry point. Physical mapping within this contig has further refined gene order within a large cluster of markers that were unresolved by linkage studies. The combined results of the genetic and physical mapping have further defined mouse and human linkage relationships on the distal end of human Chr 1, extending the synteny homology between mouse Chr 13 and human Chr 1q42–q44. MATERIALS AND METHODS Mouse genetic linkage mapping. Interspecific backcross (IB) progeny were generated by mating (SB/Le 1 M. spretus)F1 females with SB/Le males as described (Justice et al., 1990). The SB/Le strain of mice is derived from intercrosses of a stock carrying bg and sa alleles that arose at Oak Ridge National Laboratory, Oak Ridge, Tennessee in a (C3H/Rl 1 101/Rl)F1 background (Lane and Murphy, 1972, L.B. Russell, pers. comm. Oak Ridge, TN, 1996). Up to 131 N2 animals from the IB were analyzed for each marker listed in Table 1 and Fig. 1. DNA isolation, restriction endonuclease digestion, agarose gel electrophoresis, and Southern blot transfers were performed as described (Jenkins et al., 1982), except that Southern blots were prepared with MagnaCharge nylon membrane (MSI, Westboro, MA). Southern hybridizations were carried out using the method of Church and Gilbert (1984). The probes for Nid, Tcrg, Inhba, Rasl1, Fim1, Bmp6, and Srev5 have been previously described (Justice et al., 1990; Jenkins et al., 1991). Each of the probes listed in Table 1 was hybridized to Southern filters containing SB/Le or M. spretus DNAs digested with multiple restriction endonucleases to detect a restriction fragment length polymorphism (RFLP) appropriate for mapping. Each of the probes in Table 1 were gel-purified and labeled with [a-32P]dCTP using a random prime labeling kit (Stratagene, La Jolla, CA) using standard procedures, except that the probes for Gli3 and Ryr2 were labeled using 100 ng of each of the gene-specific primers substituted for the random primers of the labeling kit. DNAs from the N2 animals were digested with appropriate restriction enzymes, transferred to membranes, and hybridized with the labeled probes. Restriction enzymes and RFLP sizes are presented in Table 1. In each case , the RFLP detected in M. spretus DNA was followed in the N2 offspring. Our panel of IB progeny has been analyzed for the map locations of simple sequence length repeat polymorphisms (SSLPs) and RFLPs that were mapped previously to each of the mouse autosomes and the X chromosome (Copeland et al., 1993; Dietrich et al., 1994; Justice et al., 1990; O’Conner et al., 1996; MIT Center for Genomic Research, Database Release 1996; M. J. Justice, unpublished results). To map Chr 13-specific SSLP markers in the bg–sa interval, Mouse MapPairs (Research Genetics, Huntsville, AL) identifying the Chr 13specific SSLPs listed below (Fig. 1) were used to amplify genomic DNA from SB/Le, M. spretus, and N2 backcross mice. The sizes of SSLPs detected in SB/Le and M. spretus mice are the same as those previously reported for C3H and M. spretus (MIT Genome Center, Database Release, 1996) with the following exceptions: D13Mit133 amplified a 150-bp product in SB/Le, D13Mit10 amplified a 177-bp product in SB/Le, and D13Mit78 amplified a 240-bp product in SB/ Le. One series of SSLP markers (D13Mit1, 16, 17, 56, 57, 158, 80, 55, 44, 14, 135, 133, 38, 60, 87, and 62) was analyzed using polymerase chain reaction conditions as described (Liu et al., 1994; Bassam et al., 1991). Another series of SSLP markers (D13MIT114, 236, 215, 216, 300, 153, 240, 237, 173, 305, 271, 206, 207, 272, and 58) were analyzed per Research Genetics suggested procedures using a [g32 P]dATP radiolabeled primer and 50 ng of genomic DNA on an Idaho Technologies Thermal Cycler: 947C hot start for 20 sec, 947C for 0 sec, 557C for 0 sec, and 727C for 20 sec, for 30 cycles. Linkage and recombination distances were analyzed using the pro-

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gram MapManager Version 2.6 (Kenneth F. Manley, Roswell Park Cancer Institute, Buffalo, NY), with options for backcross calculations. Gene order is determined by minimizing the number of recombination events required to explain the allele distribution patterns. YAC isolation. The Princeton and Whitehead Mouse YAC Libraries were screened by PCR according to the distributors’ instructions. The PCR primers specific for Nid amino terminal sequences (5*CCAGCCACAGAATACCATCC-3* and 5*-GGACATACTCTGCTGCCATC-3*) were used to screen the libraries. Yeast plugs were prepared as previously described (Gnirke et al., 1993), and the sizes of the YACs were determined by pulsed-field electrophoresis on a BioRad CHEF DR2, with a pulse time of 10–100 s. YAC end isolation. All YAC ends were isolated using inverse PCR (Joslyn et al., 1991). Each resulting PCR product was gel-purified and cloned using a TA cloning kit (Invitrogen, San Diego, CA). The PCR products were sequenced using T7 and SP6 primer sites present on the plasmid. To create STSs, PCR primers specific for each end were designed and tested as described below. Somatic cell hybrid mapping of YAC ends. The primers listed below were initially tested on mouse genomic DNA to ensure that they amplified only the expected size band. These primers were then tested against a panel of mouse/hamster somatic cell hybrids. These hybrids have been previously characterized (Kozak et al., 1975), but it was necessary to test each hybrid for the presence of the bg region. This was carried out by a PCR analysis with the dinucleotide repeat markers D13Mit44 and D13Mit173, both of which map to the bg region (Figs. 1 and 2). Using this test, it was determined that 1 of 4 hybrids that we had obtained contained this region of mouse chromosome 13 (data not shown). All of the primer pairs listed in Table 1 amplified the expected size band only from the hybrid that also contained other mouse chromosome 13 markers. In some instances, Southern blots of DNA from these hybrids were probed directly with the inverse PCR products.

RESULTS

Genetic Linkage Mapping We report the order of 4 genes, 2 unique DNA probes identifying YAC ends, and 30 anonymous DNA markers relative to the two phenotypic markers bg and sa and 7 genes previously reported for this IB in the proximal region of mouse Chr 13. The genetic mapping was accomplished using genomic DNAs from the N2 progeny of a (SB/Le 1 M. spretus) 1 SB/Le backcross. We also localize the murine homolog of the Itpkb gene, which maps to the distal region of human Chr 1, to mouse Chr 1, and a related Itpkb sequence (Itpkb-rs1) to mouse Chr 4. SB/Le and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern hybridization with each of the probes listed in Table 1. At least one informative RFLP was identified for each probe. The M. spretus-specific RFLPs were followed in the backcross mice and their segregation distribution patterns (SDPs) were used to generate the maps presented in Fig. 2. The order of the loci was determined by the analysis of up to 131 N2 animals (Fig. 1). The most likely gene order and the ratio of the total number of mice carrying recombinant chromosomes to the total number analyzed for each pair of loci are: centromere– D 1 3 M i t 1–4/124–D 1 3 M i t 1 5 8–1/130–(D 1 3 M i t 8 0, D13Mit55)–1/130–(Actn2, Ryr2, Nid, D13Ut1, bg, 22B, D 1 3 U t 2, D 1 3 M i t 4 4, G l i 3)–2/130–R a s l 1–1/130–

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Anonymous DNA sequence, Utah-2

Anonymous DNA sequence, Utah-3

Glioblastoma oncogene-3 homolog Accession No. M57609

Inositol 1,4,5-trisphosphate 3-kinase B Accession No. X74227

Cardiac ryanodine receptor Accession No. J05200

D13Ut2

D13Ut3

Gli3

Itpkb

Ryr2

358F-AGGAGATGCTGGCTAACACG 377R-ATGGTCCACCANCAAGCCTC Template: mouse heart cDNA Size of product: 194 bp

1558F-CTGCCTCTGAGGAAGCTGTC 2458R-CTTGCTCCTTCTTGTCGTGG Template: mouse fibroblast cDNA Size of product: 900 bp

876F-GGATAGCACCAGATTCTCCA 1046R-CTTGCTGAAGAGCTGCTACG Template: human genomic DNA Size of product: 170 bp

21.B2-3*F-AAGCATGGCAGGGATACTTG 21.B2-3*R-CAGACTCTTTCAGATGCCTC Template: mouse genomic DNA Size of product: 176 bp

195.A8-3*F-TGCTGAGGTGAAGGTTTATG 195.A8-3*R-ACCCCAGAACTTGAGAAATAG Template: mouse genomic DNA Size of product: 217 bp

195.A8-5*F-AAACCCTGCCTCAATCAAAG 195.A8-5*R-GAGCCAAATCACATCCAACA Template: mouse genomic DNA Size of product: 255 bp

3.7-kb EcoRI cDNA fragment

93.E4-5*F-GGCCTTGGCTTCCTGAGTGT 93.E4-5*R-TTAGGTAGCTGAGTGGTGGT

722F-CGACACCATCTTGCCCTCCTCGGA 1281R-CCTTGGACTCTGTGCCCTCAT Template: mouse liver cDNA Size of product: 559 bp

Primers/probe

EcoRV

BglII

PstI

Nonec

EcoRI

EcoRI

BglII

N/A

KpnI

Enzyme

8.4, 6.9

14, 9.0, 8.0, 6.6, 5.9, 4.4

3.9

9.5

11.0

16.5, 9.4, 5.0, 4.1, 3.6, 2.7, 2.5, 1.8, 1.6, 1.4

6.4, 5.0, 4.7, 2.3

SB/Le

7.6

14, 9.0, 7.4,d 6.6, 5.9, 4.4, 3.7,e 2.6e

1.35

6.0

5.0

9.4, 9.0, 6.8, 4.9, 4.1, 2.7, 2.5, 1.8, 1.6, 1.4

5.0, 4.7, 4.4,a 2.7b 0.9b

Mus spretus

Note. The Southern blots hybridized with the Gli3 and cDNA22B probes were washed three to four times at a stringency of 11 SSCP/0.1% SDS at 657C. The Southern blots hybridized with Ryr2, D13Ut1, and D13Ut2 were washed three times in 0.21 SSCP/0.1% SDS at 657C. The Southern blots hybridized with Actn2 were washed two times in 11 SSCP/ 0.1% SDS, and one time at 0.51 SSCP/0.1% SDS at 657C. a This RFLP was a faintly hybridizing band that cosegregated with markers on mouse Chr 12. b These RFLPs cosegregated with markers on mouse Chr 13. c No RFLP was detected between SB/Le and M. spretus, but the STS was mapped to Chr 13 by somatic cell hybrids. d This RFLP was a faintly hybridizing band that cosegregated with markers on mouse Chr 4. e These RFLPs cosegregated with markers on mouse Chr 13.

Anonymous DNA sequence, Utah-1

cDNA 22B

bg

D13Ut1

93.E4-5*YAC END (Actn2 genomic sequence)

a-2 Actinin (coding sequence) Accession No. M86406

Gene name

Actn2g

Actn2

Locus or STS

Restriction fragment sizes (in kb)

Sequence Tagged Sites and Sizes of Polymorphisms Detected in SB/Le and Mus spretus Mice

TABLE 1

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FIG. 1. Segregation of alleles in 131 (SB/Le 1 Mus spretus)F1 1 SB/Le IB progeny. Each column represents the chromosome identified in the IB progeny that was inherited from the (SB/Le 1 M. spretus) F1 parent. The white boxes represent the presence of a M. spretus allele as a heterozygote, whereas the black boxes represent the presence of an SB/Le allele as a homozygote. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A subset of 18 animals exhibiting crossovers between D13Mit1 and D13Mit62 were analyzed for an additional series of markers. These animals are shown in the box labeled ‘‘interval analysis.’’ The region involved in the interval analysis is shown boxed on the left-hand side and is included underneath the main haplotype figure. These results are anchored to the genes mapped on the complete panel shown above by the locus indicated in parentheses and are also connected by lines or arrows to the chromosomes analyzed from the complete set. aEight of the animals in this row were not analyzed for D13Mit133. Seven of these animals were not analyzed for D13Mit1. bTwo of these animals were not analyzed for D13Mit133, and one animal was not analyzed for D13Mit38. cOnly one of these animals was analyzed in the interval analysis.

(D13Mit14, 135)–3/120–D13Mit133–1/120–Fim1–3/ 131–sa – 3/129 – D13Mit38 – 1/129 – D13Mit87 – 6/130 – D13Mit62. In 130 animals no recombination was observed among 9 loci in a cluster of loci that includes bg. If we were to assume that the next animal would show a crossover, we could calculate a 95% confidence limit for recombination distance within this cluster for the total of 130 animals. This 95% confidence limit would suggest that each of these loci lie within 2.2 cM of each other. This genetic distance may correlate to as much as 4.5 million basepairs. No recombination was observed in 64 animals between Srev5 and D13Mit62, suggesting that these loci lie within 4.6 cM of each other. Srev5 is included in a cluster with D13Mit62 (Fig. 2). Animals carrying certain crossovers are valuable for determining gene order in the bg–sa region. Therefore, animals carrying these crossovers were used in an interval analysis (Bell et al., 1995) to map additional loci (Fig. 1). A subset of 18 animals exhibiting crossovers between the D13Mit158 marker proximal of bg and the D13Mit38 marker distal of sa were analyzed for a number of SSLP markers that were previously placed on the proximal end of mouse Chr 13 (MIT Center for

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Genome Research, 1996; Dietrich et al., 1994; O’Conner et al., 1996). Any marker that mapped outside this interval exhibited crossovers with one or more of the two outside markers. For this analysis, an additional 19 SSLP markers were placed relative to the previously mapped gene markers and the phenotypic markers bg and sa. These analyses gave the number of crossovers and locus order: [(D13Mit158), D13Mit236, 300]–1/18– [(D 1 3 M i t 8 0 , 5 5), D 1 3 M i t 2 1 5 , 2 1 6] – 1/18 – [(bg), D13Mit173, 237, 240]–2/18 – [(Rasl1), D13Mit153, 305, 271, 207, 16, 17, 56, 57] – 1/18 – [(D13Mit135), D13Mit272, 58] – 3/18 – (D13Mit133) – 1/18 – [(Fim1), D 1 3 M i t 2 0 6] – 3/18 – (sa) – 2/18 – D 1 3 M i t 6 0 – 1/18– (D13Mit38). The markers that were mapped on the entire panel are placed in parentheses and are ‘‘anchors’’ for the interval analysis. Except for D13Mit60, each of these markers mapped to one of the previously defined clusters, and therefore, is placed on the map as a part of this cluster. The recombination distances between each cluster of genes and flanking loci were determined using the locus from each cluster that was analyzed for the most N2 animals. The only marker that mapped outside one of the previous clusters is D13Mit60, which mapped between sa and D13Mit38. For the

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FIG. 2. (A) A partial Chr 13 linkage map showing the location of markers mapped in this study. The numbers shown to the right of the chromosome are the centimorgan (cM) distances from the centromere and reflect the recombination distances between loci in our IB. Our linkage map is oriented with the consensus map at bg, which is 7 cM from the centromere. The loci that have been mapped in the human are underlined, and their location in the human is given in the center. (B) A partial comprehensive linkage map of mouse Chr 13 (Justice and Stephenson, 1996), showing only markers relevant to this study. The numbers on the left of this map are the cM distances from the centromere on the composite map, and the recombination distances between markers are an estimate based on multiple crosses involving many loci. The loci that have been mapped in the human are underlined, and their location in the human is given in the center.

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sa/D13Mit60/D13Mit38 segment, the recombination distance was estimated using proportionate recombination fractions from sa to D13Mit60 and from D13Mit60 to D13Mit38 (Bell et al., 1995). Including loci that have been previously mapped on this backcross that map to defined clusters, the recombination frequencies (expressed as genetic distance in centimorgans { standard error) between the loci are centromere–D13Mit1–3.2 { 1.6–[(D13Mit158), 236, 300]– 0.8 { 0.8–[(D13Mit80, 55), 215, 216]–0.8 { 0.8– [(Actn2, Ryr2, D13Ut1, bg, D13Mit44, 22B, Nid, D13Ut2, Gli3), D13Mit173, D13Mit237, D13Mit240]– 1.5 { 1.1–[(Rasl1, Inhba,Tcrg), D13Mit16, 17, 56, 57, 153, 207, 271, 305]–0.8 { 0.8–[(D13Mit14,135), D13Mit58, 272]–2.5 { 1.4–D13Mit133–0.9 { 0.9– [(Fim1), D13Mit206]–2.3 { 1.3–sa–1.5 { 1.5– D13Mit60–0.8 { 0.8 (D13Mit38, Bmp6)–0.8 { 0.8– D13Mit87–4.6 { 1.8–(D13Mit62, Srev5). Although the locations of Tcrg, Rasl1, Nid, Inhba, Bmp6, Fim1, and Srev5 relative to bg and sa have been previously reported for this backcross (Justice et al., 1990; Jenkins et al., 1991), the results presented in this study provide the gene order of 36 additional loci relative to the previously mapped loci in a single cross. An Actn2-related sequence mapped to mouse Chr 12 (data not shown). Mapping of the mouse homologue of rat and human inositol trisphosphate 3 kinase B (Itpkb) that maps to human Chr 1q41–q43 (Erneux et al., 1992) revealed that this gene did not map to mouse chromosome 13. Instead, Itpkb maps to mouse chromosome 1 (Fig. 3). Only 57 N2 animals from the backcross were analyzed for each of 4 markers on mouse Chr 1 (Fig. 3); however, additional animals were analyzed for some pairs of loci. The most likely gene order and the ratio of the total number of mice carrying recombinant chromosomes to the total number analyzed for each pair of loci are: D1Mit10 – 8/57 – D1Mit33 – 9/57 – Itpkb – 3/66–D1Mit17. The recombination frequencies (expressed as genetic distance in centimorgans { standard error) between the loci are centromere–D1Mit10–14.0 { 4.6–D1Mit33– 15.8 { 4.8–Itpkb–4.5 { 2.6–D1Mit17. Physical Mapping in the beige Region To begin the physical isolation of this region with the intention of identifying the gene altered in bg/bg mice, nidogen (Nid) was used as a molecular entry point to assemble a yeast artificial chromosome contig (Fig. 4). Screening the Princeton and Whitehead mouse YAC libraries with primers specific for NID amino terminal sequences resulted in the isolation of YACs C9.E7, C96.G11, 195.A8, and 151.H1. The ends of some of these YACs were isolated using inverse PCR and sequenced. The sequence was then used to create a new pair of PCR primers specific for this DNA site. These new markers are generically called sequenced tagged sites (STS) and provide a molecular marker for a given chromosomal location. Both ends of YAC 195.A8 were isolated and used to create STSs [D13Ut1(195.A8-5*)

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FIG. 3. Linkage of Itpkb to mouse Chr 1, showing homology to human Chr1q. Each column represents the chromosome identified in the IB progeny that was inherited from the (SB/Le 1 M. spretus) F1 parent. The white boxes represent the presence of a M. spretus allele, whereas the black boxes represent the presence of an SB/Le allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. Numbers to the right indicate the recombination distance between markers. Itpkb maps to a region of synteny with human Chr 1q41–q43 (Erneux et al., 1992). An Itpkbrelated sequence (Itpkb-rs1) maps to mouse Chr 4 (data not shown).

and D13Ut2(195.A8-3*)]. These STSs were localized to the bg/Nid cluster using the IB (Figs. 1 and 2). This linkage is expected since this YAC was isolated using Nid as the probe, but also demonstrates that both ends of this YAC map to the appropriate region and demonstrates that this YAC is not chimeric. To isolate more of the bg region, the ends of YAC 195.A8 [D13Ut1(195.A8-5*) and D13Ut2(195.A8-3*)] were used to rescreen the two above mentioned YAC libraries. This resulted in the isolation of six YACs using D13Ut1 (195.A8-5*) and eight YACs using D13Ut2 (195.A8-3*) (Fig. 4). Each of the YACs isolated using the STS D13Ut2 was stable and contained only one YAC band per lane. Each of the YACs isolated using the STS D13Ut1 (22.E6, 77.D3, 68.E12, 55.F3, 93.E4, and C67-C7) was unstable, because these YACs each contained multiple bands per isolate. Repeated passage

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FIG. 4. YAC Map of the bg region on mouse Chromosome 13. This YAC contig spans approximately 2.5 Mb, but the precise determination of its size is difficult due to the instability of the YACs isolated using D13Ut1 (see Results). Solid circles represent new STS, whereas open circles denote chimeric YAC ends. The position and relative size of the known genes bg, Nid, and Actn2 are depicted as open boxes. The closed squares represent SSLP markers that mapped to this YAC contig as determined by PCR from YAC DNAs.

of strains containing these six YACs would result in YACs that became smaller. Because of the instability associated with these YACs, this portion of the YAC physical map is less certain. Additional YAC ends were isolated and STS developed. The YAC end 93.E4-5* identified sequences identical to human a2-actinin (ACTN2), and Actn2 did map to the same cluster (Figs. 1, 2, and 4). We were unable to map the 21.B2-3* end (D13Ut3) genetically because it was not polymorphic on our IB panel, but this marker did map to chromosome 13 as determined by PCR analysis of the mouse/hamster somatic cell hybrids (data not shown). The markers D13Mit44 and D13Mit173 were contained on some of these YACs, but four other markers shown to map to the bg interval (i.e., Gli3, Ryr2, D13Mit237, and 240) were not contained within any of these YACs. Marker D13Mit114 is also contained within this contig but could not be genetically mapped because it would not amplify a product from M. spretus DNA. We also identify the location of known genes present within our YAC contig (Fig. 4). It should be noted that we were unable to orient our YAC contig relative to the genetic map because all molecular markers present on the YAC contig mapped to the previously mentioned cluster of markers surrounding bg (Fig. 1).

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DISCUSSION

Using an IB between SB/Le and M. spretus mice, we have developed a molecular genetic linkage map of the proximal end of mouse Chr 13. In parallel, we have developed a YAC contig of some of this region that includes bg. These studies orient multiple SSLP markers and the Gli3, Actn2, and Ryr2 genes relative to bg and sa. Actn2 was mapped to mouse Chr 13 for the first time, while Ryr2 was more precisely localized to the proximal end of mouse Chr 13. Itpkb mapped to the distal end of mouse Chr 1. Three new mouse STSs were developed from the YAC contig, and two were localized in the IB. The molecular markers mapped in this study provide a starting point for further finestructure analysis in the bg–sa region. Localization of bg within the Physical Map The YAC contig isolated in this study was developed and ultimately utilized for the identification of the gene altered in bg/bg mice (Perou et al., 1996b). The starting point for the isolation of the bg region was a marker, Nid, which had previously shown no recombination with bg (Jenkins et al., 1991). Based upon our physical

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and genetic maps, we believe that bg and Nid are very tightly linked. The extreme conserved synteny between this region of mouse chromosome 13 and human chromosome 1 suggests that the human CHS gene should lie on human chromosome 1. Support for this hypothesis comes from mapping studies of the human CHS disease trait and the human homologue of the bg gene that demonstrate that both are on human Chr 1q42– q44 (Barrat et al., 1996; Fukai et al., 1996). Additional support for this hypothesis comes from our past studies in which we demonstrated that murine YACs containing the beige gene can also complement the human CHS mutation (Perou et al., 1996a). These three results demonstrate that bg does represent the human CHS gene, as has also been reported by Barbosa et al. (1996). The physical map presented here provides a valuable tool, but also underscores the problems associated with YAC cloning. The YACs isolated using Nid and D13Ut2 were all relatively stable. Two of these YACs contained the complete wild-type bg gene as determined by in vitro complementation of the bg phenotype (Perou et al., 1996a). The YACs isolated using the STS D13Ut1, however, were all unstable and difficult to work with. Each YAC was represented by multiple bands, all of which contained similar sequences. These multiple bands presumably arose due to the deletion of YAC insert sequences in a significant fraction of cells during culture. If bg had been located on these unstable YACs, then our YAC complementation strategy used to isolate the wild-type counterpart of bg may not have succeeded. The cause of the YAC instability was not determined. Comparison with Previous Mapping Results Of the gene markers mapped in this study, Actn2 and Itpkb had not been previously placed on a mouse chromosome. An Actn2-related sequence (Actn2-rs1) mapped to mouse Chr 12 to a region syntenic with human Chr 14q (data not shown). Notably, human ACTN1 maps to Chr 14q22–q24 (Youssoufian, et al., 1990); therefore, it is possible that this weakly hybridizing sequence identifies the mouse homologue of ACTN1. Ryr2 was previously mapped using in situ hybridization analysis to proximal mouse Chr 13, bands A1–A2 (Mattei et al., 1994). Its localization to the bg/ Nid cluster is not surprising, since it also maps to human Chr 1q42.1–q43 (Otsu et al., 1993). None of the SSLP markers have been mapped directly with bg; however, since D13Mit44 identifies Nid, our results can be compared with the genetic linkage map published by the genome groups at MIT. The order and approximate recombination distance (in cM) of markers that have been mapped by the Genome Group at MIT markers and were included in our study are (D13Mit16, 55)–5–(D13Mit158, 300, 236, 1)–1– D13Mit206–0.5–(D13Mit44, 238, 114, 173, 127, 240, 56, 57, 271, 207, 80, 133)–1–(D13Mit17, 135, 272, 14)– 0. 5 – D 1 3 M i t 5 8 – 3 . 6 – D 1 3 M i t 6 0 – 0 . 5 – D 1 3 M i t 3 8

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(Mouse Genome Database, 1996; MIT Center for Genome Research, 1996). Markers that exhibit discrepancies in gene order with our mapping results are underlined. D13Mit1 is the most obvious of these, because it is the most proximal of our markers. The proximal location of D13Mit1 is also consistent with another cross (R. P. Elliot, pers. comm., Buffalo, NY, 1996). However, D13Mit16, D13Mit55, and D13Mit206 lie much more distal on our map compared with the MIT map (Fig. 2). These data should be taken into consideration when assembling the physical map of proximal mouse Chr 13. Our results break up a large cluster of markers on the MIT map into two segments, one located more proximally containing Nid (D13Mit44), bg, D13Mit114, 173, 238, 237, and 240 and one distal containing D13Mit56, 57, 271, and 207. D13Mit133 is placed more distal on our map than on the MIT map. These differences are not surprising considering the small number of mice analyzed in the MIT intercross and that discrepancies in the MIT backcross map are only currently being resolved (MIT Center for Genome Research, 1996). Correlation of the Genetic and Physical Maps One outcome of our combined physical and genetic maps is to order many genes in a single cross and to order several markers at a fine-structure level (Figs. 2 and 4). The genetic linkage studies involved only 131 IB animals and therefore did not provide a fine resolution of all relevant markers. Although the YACs isolated in this study span approximately 2.5 Mb, neither end of the YAC contig identified a crossover that would orient the contig within the genetic map. The cluster of markers surrounding bg likely lies within a 2.2-cM genetic interval. If genetic distance is strictly correlated with physical distance in this interval, we expected the contig to be oriented. Therefore, our physical map provides the order of Actn2, D13Mit114, D13Mit173, D13Ut1, D13Ut2, bg (22B) and Nid, but cannot order these markers within the framework of the markers that were mapped genetically. Note that no recombination was observed between Gli3 and Nid in this backcross, although recombination in other crosses has placed Gli3 distal to bg (Lane, 1971; Lyon et al., 1967). This observation, combined with the homology breakpoint between human Chr 1q43 and 7p13, allows us to conclude that Gli3 is distal to Nid and bg. Genes present on our YAC contig all mapped to human Chr 1 (this study and data not shown). Therefore, we did not cross over the synteny breakpoint between human Chr 1 and human Chr 7 on mouse Chr 13. Since Gli3 did not recombine with Nid on this IB, it is not surprising that the physical map could not be oriented within the genetic map. A Comparative Map of Human Chr 1q42–q44 Most of human Chr 1q is conserved on mouse Chr 1 (Seldin, 1996); however, linkages on the distal end are

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FIG. 5. A comparative map of human Chr 1q41–q44. Banding patterns for the distal portion of human Chr 1q are shown. Genes that have been mapped in mouse and human are shown in the middle and include H2.0 (Drosophila)-like homeo box-1 (HLX1), peptidase C (PepC), ITPKB, skeletal muscle, alpha-1 actin (ACTA1), angiotensinogen (AGT), RYR2, ACTN2, NID, CHS (bg), and Primase polypeptide 1 (PRIM1) (OMIM, 1996; this study). The boxes on the right show the conserved linkages on mouse Chrs 1 (open box), Chr 8 (black on white box), and Chr 13 (white on black box), and one locus mapping to Chr 10 (indicated as a circle).

fragmented on mouse Chr’s 1, 8, 13, and 10 (Online Mendelian Inheritance in Man, 1996). In an attempt to clarify linkage relationships on the distal end of human Chr 1, we localized several genes that were known to map to the human Chr 1q42–q44. These included Actn2, Ryr2, and Itpkb (Beggs et al., 1992; Otsu et al., 1990, Erneux et al., 1992). Notably, Actn2 and Ryr2 map to mouse Chr 13, with Actn2 contained within the Nid-based YAC contig. Ryr2 is in the bg cluster on the genetic map, but does not lie on our YAC contig (data not shown). Itpkb maps to mouse Chr 1 in a region that shows homology with human Chr 1q41–q43 (Seldin, 1996; Mouse Genome Database, 1996). We propose that detailed genetic linkage maps in the mouse may refine gene order in human. Our studies combined with human mapping results allow a prediction of gene order on distal human Chr 1 (Fig. 5) and suggest that markers closest to human band 1q43 map to mouse Chr 13. Further, our studies predict that genes that map to bands 1q41–q43 in human and to Chr 1 in mouse must lie more proximal in human, near 1q41–q42.1, whereas genes that map to mouse Chr 13 must lie more distal, near 1q43 (Fig. 5). A small fragment of the chromosome between 1q41–q42.1 and 1q43 maps to mouse Chr 8 (Fig. 5). Still, additional genes that remain to be mapped in the mouse have been mapped to human Chr 1q42–q44 (Online Mendelian Inheritance in Man, 1996). Our results have extended the synteny homology between mouse Chr 13 and human Chr 1 and between mouse Chr 1 and human Chr 1. Further Studies of the beige–satin Region The genetic linkage map developed in this study is not a fine-structure linkage map, but has been very useful for localizing genes within cluster regions accessible by physical maps. Complementation assays in

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these studies were essential for narrowing down the physical region that contained bg (Perou et al., 1996a); however, further studies of this interval may require a finer mapping strategy. The markers ordered in this study provide a strategy for developing a fine-structure map of the bg–sa interval. We are currently selecting for crossovers between bg and D13Mit38, which is distal to sa, to develop reagents for a fine-structure map of the entire interval. Crossovers identified in these animals will be useful for the molecular identification of the gene encoding sa. A number of intriguing loci that are conserved between mouse and human lie within the bg–sa interval, including many expressed sequences, potential models of human disease, and several gene clusters (Justice and Stephenson, 1996). However, there are relatively few mutations compared with the number of genes. With the progress of the human genome project, it is likely that new genes will continue to be identified within this interval. A future goal is to examine the biological function of these genes. We propose that the physical map created in this study, as well as other overlapping physical maps in the bg–sa interval, will be useful in a dissection of gene function using chemical mutagenesis in screens utilizing deletions (Tease and Fisher, 1993; Ramirez-Solis et al., 1995) and recessive visible markers (Rinchik, 1991). ACKNOWLEDGMENTS Mouse/hamster somatic cell hybrids were kindly provided by Dr. C. A. Kozak, National Institutes of Health. We thank Drs. Dabney Johnson, Michael Mucenski, and David Garfinkel for their critical comments on the manuscript. M.J.J. is supported by the U.S. Department of Energy, under Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corp., by the National Institutes of Health, 7R29CA63229-02, and by an award from the American Cancer Society, JFRA-553. A.P. is the recipient of a Goldwater Scholarship. J.K. is supported by the National Institutes of Health, HL26922, and C.M.P. is the recipient of NIH Genetics Training Grant T32GM07464.

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